catalysts

Article Bacillus subtilis A—Lipase or ?

Paula Bracco 1, Nelleke van Midden 1, Epifanía Arango 1, Guzman Torrelo 1, Valerio Ferrario 2, Lucia Gardossi 2 and Ulf Hanefeld 1,* 1 Biokatalyse, Afdeling Biotechnologie, Technische Universiteit Delft, Van der Maasweg 9, 2629 HZ Delft, The Netherlands; [email protected] (P.B.); [email protected] (N.v.M.); [email protected] (E.A.); [email protected] (G.T.) 2 Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Trieste, Via Licio Giorgieri 1, 34127 Trieste, Italy; [email protected] (V.F.); [email protected] (L.G.) * Correspondence: [email protected]; Tel.: +31-15-278-9304

 Received: 20 February 2020; Accepted: 5 March 2020; Published: 7 March 2020 

Abstract: The question of how to distinguish between and is about as old as the definition of the subclassification is. Many different criteria have been proposed to this end, all indicative but not decisive. Here, the activity of lipases in dry organic solvents as a criterion is probed on a minimal α/β fold , the Bacillus subtilis lipase A (BSLA), and compared to antarctica lipase B (CALB), a proven lipase. Both show activity in dry solvents and this proves BSLA to be a lipase. Overall, this demonstrates the value of this additional parameter to distinguish between lipases and esterases. Lipases tend to be active in dry organic solvents, while esterases are not active under these circumstances.

Keywords: hydrolase; lipase; esterase; Bacillus subtilis lipase A; transesterification; organic solvent; water activity

1. Introduction Lipases and esterases both catalyze the of . This has led to the longstanding question: how can we distinguish between a lipase and an esterase? As the simple hydrolysis of an does not suffice, a range of different criteria has been suggested [1–5]. (1) The oldest distinction is the kinetic and structural criterion of interfacial activation, which was already described in 1936 [6]. However, several lipases do not fulfill this; in particular, the much-used Candida antarctica lipase B (CALB) does not [7]. (2) Directly linked to the interfacial activation is the lid that covers the of many lipases and, via a conformational change, makes the active site more accessible once an interface is present. Again, this is not the case for all lipases [1–3,7,8]. (3) Primary sequence data were shown not to be distinctive enough [2]. (4) Substrates and inhibitors, such as , can be utilized to distinguish between esterases and lipases but, again, they are not precise. However, the different ranges are indicative. Esterases tend to be capable of the hydrolysis of water-soluble esters and, in general, short and/or branched side chain esters, while lipases hydrolyze , apolar esters, substituted with linear side chains, as well as waxes. This is seen as a reliable but not decisive criterion [2,9]. (5) The activity of the enzyme in the presence of (water-miscible) organic solvents has been proposed as a property of lipases, but other fulfill this criterion, too [2,10–14]. (6) A parameter already investigated some time ago is the activity of lipases in the absence of water, i.e., in modestly polar, water-non-miscible solvents at very low water activities (aw). Out of all enzymes tested, only lipases and the closely related are active at low aw [1–5,10,15–19]. While not all lipases display this property, it is highly distinctive [20,21]. To probe whether aw is indeed a suitable parameter to distinguish between lipases and esterases and between lipases and other hydrolases in general, we studied the behavior of Bacillus subtilis

Catalysts 2020, 10, 308; doi:10.3390/catal10030308 www.mdpi.com/journal/catalysts Catalysts 2020, 10, 308 2 of 17 lipase A (BSLA) [9]. BSLA is a small (181 amino , 19 kDa) hydrolase (Figure1). It is Catalysts 2019, 9, x FOR PEER REVIEW 2 of 17 neither interfacially activated nor does it have a lid (criteria one and two) [9,22–24] and sequence datainterfacially are not conclusive, activated nor but does it is ait minimalhave a lidα (crite/β hydrolaseria one and fold two) enzyme [9,22–24] [9 and,23,25 sequence]. The substrate data are not range clearlyconclusive, qualifies but BSLA it is a as minimal a lipase, α/ asβ hydrolase does the stabilityfold enzyme in the [9,23,25 presence]. The of substrate solvents range [9,26 –clearly29]. This stabilityqualifies has BSLA even as been a lipase, significantly as does the improved stability inin recentthe presence mutational of solvents studies [9,26–29]. and BSLA This mutantsstability has can be veryeven stable been in significantly the presence improved of water-miscible in recent solvents,al studies such as and dimethyl BSLA mutants sulfoxide can (DMSO), be very stable dioxane andin trifluoroethanol the presence of [30water-miscible,31]. Studies solvents, on BSLA su inch dry as organicdimethyl solvents sulfoxide are, (DMSO), however, dioxane missing. and As an experimentaltrifluoroethanol parameter, [30,31]. Studies we demonstrate on BSLA in thedry activityorganic ofsolvents BSLA are, in dryhowever, toluene. missing. Toluene As an is not water-miscibleexperimental andparameter, has a logPwe demonstrate of 2.5 [32]. the It isactivity commonly of BSLA used in dry in organictoluene. Toluene synthesis is andnot water- is highly suitablemiscible for and lipases has a and logP also of 2.5 other [32]. enzymesIt is commonly with used an α /inβ organichydrolase synthesis fold. and To date, is highly only suitable lipases for were lipases and also other enzymes with an α/β hydrolase fold. To date, only lipases were shown to be shown to be active in toluene with a very low aw [1,3]. active in toluene with a very low aw [1,3].

FigureFigure 1. Bacillus1. Bacillus subtilis subtilislipase lipase A A (BSLA) (BSLA) isis the smalle smallestst serine hydrolase with with an anα/βα hydrolase/β hydrolase fold. fold. WithWith only only 181 181 amino amino acids, acids, it it has has a a molecular molecular weightweight of 19 19 kDa. kDa. The The depicted depicted BSLA BSLA structure structure is pdb is pdb 1R501R50 and and the the catalytic triad His156, His156, Ser77Ser77 andand Asp133 and and the the oxyanion hole Ile12 Ile12 and and Met78 Met78 are are highlighted.highlighted. The The figure figure was was created created with with PyMOL.PyMOL.

Additionally,Additionally, we we extend extend the the structural structural assignment assignment of of the the hydrolase hydrolase character character with with the the GRID-based GRID- (Fortranbased program(Fortran program [33]) Global [33]) Global Positioning Positioning System System in Biological in Biological Space Space (Bio (Bio GPS) GPS) investigation investigation [ 33]. BioGPS[33]. utilizesBioGPS surfaceutilizes shapesurface and shape polarity and aspolarity criteria. as Itcriteria. is neither It is based neither on based direct on sequence direct sequence comparison, comparison, nor on structure superimposition [33,34]. Earlier studies with this method had placed nor on structure superimposition [33,34]. Earlier studies with this method had placed CALB in both CALB in both the esterase and lipase group. CALB works extremely well in dry solvents and is, the esterase and lipase group. CALB works extremely well in dry solvents and is, therefore, often therefore, often applied in reactions that require these conditions, such as dynamic kinetic resolutions applied in reactions that require these conditions, such as dynamic kinetic resolutions [1,3,7,35]. On [1,3,7,35]. On the other hand, it misses interfacial activation and major conformational changes do not thetake other place hand, when it misses CALB comes interfacial into contact activation with andan apolar major second conformational phase (see above). changes As do such, not BioGPS take place whenrecognized CALB comes the ambivalence into contact in with the assignment an apolar secondof CALB phase as a lipase (see above). well. As such, BioGPS recognized the ambivalenceHere, we indescribe the assignment the investigation of CALB of asBSLA a lipase by BioGPS well. and a comparison to other lipases, in particularHere, we CALB. describe We the also investigation probe the lipase of BSLA character by BioGPS of both and BSLA a comparison and CALB to otherat different lipases, aw in. In particular this CALB.manner, We also new probe experimental the lipase and character computational of both criteria BSLA andfor the CALB esterases at different and lipases aw. In are this introduced manner, new experimentaland investigated. and computational criteria for the esterases and lipases are introduced and investigated.

2. Results2. Results

2.1.2.1. BioGPS BioGPS BioGPSBioGPS descriptors descriptors can can be be utilized utilized toto exploreexplore en enzymezyme active active site site properties properties and and to group to group them them accordingaccording to theirto their similarities similarities and and diff erences.differences. As such,As such, they they can helpcan tohelp explore to explore promiscuous promiscuous activities. activities. In an earlier study, the character of CALB was investigated in a set of 42 serine hydrolases.

Catalysts 2020, 10, 308 3 of 17

In anCatalysts earlier 2019 study,, 9, x FOR the PEER character REVIEW of CALB was investigated in a set of 42 serine hydrolases. The3 of 17 set contained 11 , nine , 11 esterases and 11 lipases, one of them being CALB [33]. Here, weThe expand set contained this set with11 amidases, BSLA, utilizing nine proteases, the pdb 11 1R50esterases with and a resolution 11 lipases, ofone 1.4 of Åthem for thebeing structural CALB information[33]. Here, (Table we expand S1). Three this set probes with BSLA, were used utilizing to map the specificpdb 1R50 electrostatic with a resolution and geometrical of 1.4 Å for active the sitestructural properties. information The O-probe (Table evaluates S1). Three the H-bond probes donor were properties used to ofmap the specific enzymes; electrostatic the N1 probe, and on thegeometrical contrary, evaluates active site the properties. H-bond acceptor The O-probe properties evaluates and the the DRY H-bond probe evaluatesdonor properties the hydrophobic of the interactionsenzymes; [the33]. N1 The probe, DRY on probe the contrary, is clearly evaluates of special the importance H-bond acceptor for enzymes properties that and accept the hydrophobicDRY probe substrates,evaluates as the is thehydrophobic case for lipases. interactions [33]. The DRY probe is clearly of special importance for enzymesConsidering that accept each hydrophobic property separately, substrates, the as is O-probe the caselocated for lipases. BSLA (pdb 1R50) not among the lipases,Considering but in the amidases each property cluster, separately, together the with O- aprobe number located of esterases BSLA (pdb (Figure 1R50) S1a). not among Equally, the the lipases, but in the amidases cluster, together with a number of esterases (Figure S1a). Equally, the N1-probe (Figure S1b) placed BSLA among the amidases. The DRY probe (Figure S1c), again, placed N1-probe (Figure S1b) placed BSLA among the amidases. The DRY probe (Figure S1c), again, placed BSLA amongst the amidases and esterases. This is, in all cases, in contrast to CALB (pdb 1TCA) but it BSLA amongst the amidases and esterases. This is, in all cases, in contrast to CALB (pdb 1TCA) but should also be noted that Candida rugosa lipase (CRL), a classic lipase with a prominent movement of it should also be noted that Candida rugosa lipase (CRL), a classic lipase with a prominent movement the lid (criteria one and two) is also always outside the lipase cluster in the different analyses. The of the lid (criteria one and two) is also always outside the lipase cluster in the different analyses. The previousprevious study study ascribed ascribed this this behavior behavior to to the the lower lower hydrophobichydrophobic nature of of the the active active site site of of CRL CRL (pdb (pdb 1CRL)1CRL) when when compared compared to to the the other other lipases lipases [ 8[8,33].,33]. In theIn the global global score, score, which which considers considers all all the the mappedmapped properties of of the the BioGPS BioGPS together, together, BSLA BSLA can can be foundbe found firmly firmly among among the the amidases amidases and and esterases esterases (Figure (Figure2), 2), while while CALB CALB is inis thein the lipase lipase cluster cluster in in the areathe overlapping area overlapping with the with esterase the esterase cluster. cluster. CRL, again,CRL, again, is outside is outside the lipase the lipase cluster cluster and indeedand indeed seems to takeseems a separateto take a separate position. position.

FigureFigure 2. BioGPS2. BioGPS of 43of 43 serine serine hydrolases, hydrolases, for for BSLA BSLA the the data data of pdbof pdb 1R50 1R50 were were utilized utilized (global (global score). score). Each analyzedEach analyzed enzyme structureenzyme isstructure placed withinis placed a multidimensional within a multidimensional space. Relative space. distances Relative between distances each enzymebetween and each all the enzyme other enzymesand all the are other determined enzymes by are a determined statistical principal by a statistical component principal analysis. component The pdb codesanalysis. of the The processed pdb codes enzyme of the structures processed are enzyme indicated structures in different are indicated colors according in different to colors their class: according lipases in blue,to their amidases class: lipases in red, in proteases blue, am inidases cyan andin red, esterases proteases in green; in cyan the and BSLA esterases structure in isgreen; in black. the BSLA structure is in black.

Catalysts 2020, 10, 308 4 of 17

Catalysts 2019, 9, x FOR PEER REVIEW 4 of 17 Catalysts 2019, 9, x FOR PEER REVIEW 4 of 17 BSLA is, according to its substrate range, very clearly a lipase and not an esterase. BSLA is, according to its substrate range, very clearly a lipase and not an esterase. Amidase activityBSLA has is, to according date not been to its reported substrate for range, BSLA. very While clearly initially a lipase surprising, and not these an resultsesterase. also Amidase indicate activity has to date not been reported for BSLA. While initially surprising, these results also indicate activitythat the has study to date should not be been extended reported with for an BSLA. activity Wh assayile initially for amidases. surprising, these results also indicate that the study should be extended with an activity assay for amidases. that the study should be extended with an activity assay for amidases. 2.2. Amidase Activity 2.2. Amidase Activity 2.2. AmidaseTo probe Activity for amidase activity in BSLA, an amidase activity assay is utilized (Scheme1). To probe for amidase activity in BSLA, an amidase activity assay is utilized (Scheme 1). This ThisTo assay probe employs for amidase benzyl activity chloroacetamide in BSLA, an asamidase standard activity amide. assay Theis utilized released (Scheme amine 1). reacts This assay employs benzyl chloroacetamide as standard amide. The released amine reacts with 4-nitro-7- assaywith 4-nitro-7-chloro-benzo-2-oxa-1,3-diazole,employs benzyl chloroacetamide as standard yielding amide. an The adduct released that amine can directly reacts with be quantified4-nitro-7- chloro-benzo-2-oxa-1,3-diazole, yielding an adduct that can directly be quantified chloro-benzo-2-oxa-1,3-diazole,spectrophotometrically at 475 nm [yielding36]. an adduct that can directly be quantified spectrophotometrically at 475 nm [36]. spectrophotometrically at 475 nm [36].

SchemeScheme 1. 1. AmidaseAmidase activity activity assay assay [36]. [36]. The The assay assay can can be be quantified quantified spectrophotometrically. spectrophotometrically. BSLA BSLA Scheme 1. Amidase activity assay [36]. The assay can be quantified spectrophotometrically. BSLA showedshowed no no activity activity in in this this as assay,say, ruling out amidase activity. showed no activity in this assay, ruling out amidase activity. BSLABSLA showed showed no no activity activity in in this this assay. assay. A contro A controll experiment experiment with with another another serine serine hydrolase, hydrolase, the BSLA showed no activity in this assay. A control experiment with another serine hydrolase, the acyltransferasethe from from MycobacteriumMycobacterium smegmatis smegmatis (Ms(MsAct),Act), was was performed. performed. This This enzyme enzyme is is an an acyltransferase from Mycobacterium smegmatis (MsAct), was performed. This enzyme is an acyltransferaseacyltransferase [37,38] [37,38] and and displays displays promiscuous promiscuous amidase amidase activity activity [39,40]. [39,40]. MsMsActAct exhibited exhibited activity activity acyltransferase [37,38] and displays promiscuous amidase activity [39,40]. MsAct exhibited activity inin this this 24 24 h h assay assay (> (> 20%20% conversion conversion of of the the 5 5 mM mM substrate), substrate), showing showing that that even even minor, minor, promiscuous promiscuous in this 24 h assay (> 20% conversion of the 5 mM substrate), showing that even minor, promiscuous activitiesactivities are are detectable. detectable. This This rules rules out out amidase amidase ac activitytivity for for BSLA BSLA and and supports supports the the earlier earlier assignment assignment activities are detectable. This rules out amidase activity for BSLA and supports the earlier assignment ofof the the enzyme enzyme as as a a lipase. lipase. of the enzyme as a lipase. 2.3. BSLA Activity in Dry Organic Solvents 2.3. BSLA Activity in Dry Organic Solvents 2.3. BSLA Activity in Dry Organic Solvents To probe the activity of BSLA at low aw, toluene was used as the solvent and the transesterification To probe the activity of BSLA at low aw, toluene was used as the solvent and the of 1-octanolTo probe with the vinyl activity acetate of was BSLA performed at low asa aw,test toluene reaction was (Scheme used 2as). Thethe usesolvent of 1-octanol and the as of 1-octanol with vinyl acetate was performed as a test reaction (Scheme 2). The transesterificationa long chain aliphatic of 1-octanol compound with is avinyl good acetate substrate was for performed lipases [1 as–5 ,a9 ]test and reaction vinyl acetate (Scheme is a 2). readily The use of 1-octanol as a long chain aliphatic compound is a good substrate for lipases [1–5,9] and vinyl useavailable of 1-octanol and widely as a long utilized chain acyl aliphatic donor in compound lipase catalyzed is a good acylation substrate reactions for lipases [1,3,41 [1–5,9],42]. All and reactions vinyl acetate is a readily available and widely utilized acyl donor in lipase catalyzed acylation reactions acetatewere performed is a readily with available lyophilized and BSLA. widely In utilized parallel, acyl CALB donor was alsoin lipase tested catalyzed to ensure directacylation comparability reactions [1,3,41,42]. All reactions were performed with lyophilized BSLA. In parallel, CALB was also tested to [1,3,41,42].with one of All the reactions most-used were lipases. performed CALB with was lyophilized utilized both BSLA. as lyophilized In parallel, enzyme CALB was and also immobilized tested to ensure direct comparability with one of the most-used lipases. CALB was utilized both as lyophilized ensureas Novozym direct comparability 435. The latter with preparation one of the is most-used most commonly lipases. employed, CALB was both utilized in the both laboratory as lyophilized and on enzyme and immobilized as Novozym 435. The latter preparation is most commonly employed, both enzymeindustrial and scale immobilized [43]. as Novozym 435. The latter preparation is most commonly employed, both in the laboratory and on industrial scale [43]. in the laboratory and on industrial scale [43].

Scheme 2. Test reaction for the activity of BSLA at low aw. The reaction was performed in toluene at SchemeScheme 2. 2. TestTest reaction for thethe activityactivity ofof BSLABSLA at at low low a waw.. The The reaction reaction was was performed performed in in toluene toluene at at 30 30 °C, with a ratio of 1-octanol to vinyl acetate of 1:5 and aw < 0.1, 0.23 and 0.75. 30◦C, °C, with with a ratioa ratio of of 1-octanol 1-octanol to to vinyl vinyl acetate acetate of of 1:5 1:5 and and aw a

Catalysts 2020, 10, 308 5 of 17 Catalysts 2019, 9, x FOR PEER REVIEW 5 of 17

Figure 3. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels of purified Figure 3. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels of purified BSLA (19 kDa) and Candida antarctica lipase B (CALB) (33 kDa). BSLA (19 kDa) and Candida antarctica lipase B (CALB) (33 kDa).

Three different aw were tested < 0.1 to establish whether BSLA shows the activity in dry solvent Three different aw were tested < 0.1 to establish whether BSLA shows the activity in dry solvent only observed for lipases, with aw = 0.23 as a low value at which most enzymes lose all their activity only observed for lipases, with aw = 0.23 as a low value at which most enzymes lose all their activity and aw = 0.75, an activity at which most enzymes are active [10,21,45,46]. To rigorously ascertain these valuesand awof = 0.75, the solvent an activity and reagents,at which includingmost enzymes the internal are active standard, [10,21,45,46]. decane To and rigorously the enzyme ascertain preparations these werevalues equilibrated of the solvent via the and vapor reagents, phase with including dried molecular the internal sieves standard, (activated decane at elevated and temperatures,the enzyme preparations were equilibrated via the vapor phase with dried molecular sieves (activated at elevated 5 Å) for aw < 0.1 [47]. For the other aw, the enzyme preparations and the other components were temperatures, 5 Å) for aw < 0.1 [47]. For the other aw, the enzyme preparations and the other equilibrated via the gas phase with an oversaturated solution of potassium acetate (aw = 0.23) and components were equilibrated via the gas phase with an oversaturated solution of potassium acetate sodium chloride (aw = 0.75) [48–52]. For all components, the water content was determined by Karl Fischer(aw = 0.23) titration and andsodium equilibrations chloride (a werew = 0.75) considered [48–52]. complete For all whencomponents, no changes the werewater observed content was any moredetermined (24–48 by h, TableKarl Fischer1). As vinyl titration acetate and wasequilibrations found to negatively were consi aderedffect thecomplete Karl Fischer when no titration, changes it waswere freshly observed distilled any more and dried (24–48h, with Table activated 1). As molecular vinyl acetate sieves was for found 16 h before to negatively use. The affect activity the of Karl the diFischerfferent titration, enzyme it preparations was freshly wasdistilled also followedand dried with with the activated tributyrin molecular and p-nitrophenol sieves for 16 acetate h before activity use. assaysThe activity [2,5,53 –of56 ]the during different equilibration, enzyme topreparatio establishns optimal was also equilibration followed times. with Forthe BSLA,tributyrin a small and loss p- ofnitrophenol activity over acetate time activity was observed, assays [2,5,53–56] while both during CALB preparationsequilibration, were to establish stable. optimal equilibration times. For BSLA, a small loss of activity over time was observed, while both CALB preparations were

stable.Table 1. Equilibration to different aw via vapor phase over a saturated solution of salt [47,51] and via the salt pair method [50]. All reaction components, except the acyl donor, were mixed and equilibrated

overnightTable 1. Equilibration at 30 ◦C. Finally, to different dried andaw via freshly vapor distilled phase over vinyl a saturated acetate was solution added of in salt order [47,51] to start and thevia reaction.the salt Thepair watermethod content [50]. was All determined reaction compon by Karlents, Fischer except Titration the afteracyl 48donor, h. were mixed and equilibrated overnight at 30 °C. Finally, dried and freshly distilled vinyl acetate was added in order toa startw theAgent reaction. (Vapor The Phasewater content or Salt Pair)was dete Molesrmined of by H Karl2O/mol Fischer of Salt Titration Water after Content48 h. (ppm) <0.1 Mol. sieves 0 ~20 a0.25w Agent NaAc(vapor anhydr. phase (saltor salt pair) pair) Moles of H2O/mol 1.5 of salt Water content ~180 (ppm) < 0.570.1 Na2HPO Mol.4 anhydr. sieves (salt pair) 5.00 ~ ~360 20 0.250.23 NaAc KAc anhydr. (vapor (salt phase) pair) NA 1.5 a) ~~120 180 a) 0.570.75 Na2HPO NaCl4 anhydr. (vapor phase)(salt pair) NA 5.0 ~~400 360 0.23 KAc (vapor phase) a) Not applicable (NA). NAa) ~ 120 0.75 NaCl (vapor phase) NAa) ~ 400 Once reagents and enzymes werea) equilibrated, Not applicable the (NA). reactions were performed with 100 mM 1-octanol and 500 mM vinyl acetate in previously equilibrated toluene at 30 ◦C and 1000 rpm (Figure4). Once reagents and enzymes were equilibrated, the reactions were performed with 100 mM 1- Equal activity of the enzymes (Units) was utilized as determined with the tributyrin activity assay. CALB octanol and 500 mM vinyl acetate in previously equilibrated toluene at 30 °C and 1000 rpm (Figure and, in particular, the well-established commercial preparation of CALB, Novozym 435, performed 4). Equal activity of the enzymes (Units) was utilized as determined with the tributyrin activity assay. very well. In both cases, full conversion to 1-octyl acetate was observed. In comparison, BSLA CALB and, in particular, the well-established commercial preparation of CALB, Novozym 435, displayed lower conversions (Figure4). However, the key indicator for a lipase is its activity at low performed very well. In both cases, full conversion to 1-octyl acetate was observed. In comparison, aw. Here, BSLA and Novozym 435 performed best. For the synthesis of 1-octyl acetate, the trend is a BSLA displayed lower conversions (Figure 4). However, the key indicator for a lipase is its activity at reduction in specific rate at higher aw (Figure5). BSLA is very active in dry solvent, as is Novozym low aw. Here, BSLA and Novozym 435 performed best. For the synthesis of 1-octyl acetate, the trend 435. Both display lower activities at higher aw. CALB does not follow this trend. is a reduction in specific rate at higher aw (Figure 5). BSLA is very active in dry solvent, as is Novozym 435. Both display lower activities at higher aw. CALB does not follow this trend.

Catalysts 2019, 9, x FOR PEER REVIEW 6 of 17 Catalysts 2020, 10, 308 6 of 17 Catalysts 2019, 9, x FOR PEER REVIEW 6 of 17

Figure 4. Activity of BSLA, CALB and Novozym 435 in toluene with different aw. U = µmol butyric Figure 4. Activity of BSLA, CALB and Novozym 435 in toluene with different aw.U = µmol butyric −1 x min1 in tributyrin activity assay, 0.5–1.2 U of catalyst, 1-octanol (100 mM), vinyl acetate (5 eq.), acidFiguremin 4. −Activityin tributyrin of BSLA, activity CALB assay, and 0.5–1.2Novozy Um of 435 catalyst, in toluene 1-octanol with (100different mM), a vinylw. U = acetate µmol (5butyric eq.), ISTD:× Decane−1 (500 mM), 1 mL reaction volume, 24 h, 30 °C and 1000 rpm. Blanks were performed in ISTD:acid x Decane min in (500 tributyrin mM), 1activity mL reaction assay, volume,0.5–1.2 U 24 of h,cata 30lyst,◦C and1-octanol 1000 rpm.(100 mM), Blanks vinyl were acetate performed (5 eq.), the absence of enzyme and showed no conversion. Final conversions are given as inset; the color inISTD: the absence Decane of(500 enzyme mM), and1 mL showed reaction no volume, conversion. 24 h, 30 Final °C conversionsand 1000 rpm. are Blanks given were as inset; performed the color in corresponds to the aw. correspondsthe absence toof the enzyme aw. and showed no conversion. Final conversions are given as inset; the color corresponds to the aw.

) BSLA -1 Novozym 435 ) BSLA x U x

-1 14 -1 NovozymCALB 435

x U x 14

-1 12 CALB 12 10 10 8 8 2 mol 1-octyl acetate x min 1-octyl acetate mol

(μ 2 mol 1-octyl acetate x min 1-octyl acetate mol (μ

0

Specific rate Specific aw < 0,1 aw 0,23 aw 0,75 0

Specific rate Specific aw < 0,1 aw 0,23 aw 0,75 aw

aw Figure 5. Activity of BSLA, CALB and Novozym 435 in toluene with different aw. Reaction conditions: Figure 5. Activity of BSLA, CALB and Novozym 435 in toluene with different aw. Reaction conditions: 0.5–1.2 U of catalyst, 100 mM 1-octanol, 500 mM vinyl acetate, 500 mM decane (ISTD), in dry toluene 0.5–1.2 U of catalyst, 100 mM 1-octanol, 500 mM vinyl acetate, 500 mM decane (ISTD), in dry toluene (1Figure mL reaction) 5. Activity at 30of BSLA,C and CALB 1000 rpm. and Novozym U: µmol butyric 435 in toluene acid min with1 different. Blanks wereaw. Reaction performed conditions: in the (1 mL reaction) at 30◦ °C and 1000 rpm. U: µmol butyric acid× x min− −1. Blanks were performed in the absence0.5–1.2 U of of enzyme catalyst, and 100 showed mM 1-octanol, no conversion. 500 mM vinyl acetate, 500 mM decane (ISTD), in dry toluene (1absence mL reaction) of enzyme at 30 and °C showedand 1000 no rpm. conversion. U: µmol butyric acid x min−1. Blanks were performed in the Inabsence an earlier of enzyme study, and it had showed been no demonstrated, conversion. for different CALB preparations, that this change in activityIn an in earlier the synthesis study, it reactionhad been to demonstrated, 1-octyl acetate for can different be due CALB to the hydrolysispreparations, of thethat acyl this donorchange in activity in the synthesis reaction to 1-octyl acetate can be due to the hydrolysis of the acyl donor vinyl acetateIn an earlier [47]. Therefore,study, it had the been synthesis demonstrated, reaction at for aw different< 0.1 was CALB repeated preparations, for BSLA with that athis 1-octanol change toinvinyl vinyl activity acetate acetate in the[47]. ratio synthesis Therefore, of 1:1 (Figurereaction the synthesis6 ).to Almost1-octyl reaction theacetate same at canaw rate < be0.1 anddue was conversionto repeated the hydrolysis for was BSLA observed of with the acyl a as1-octanol withdonor to vinyl acetate ratio of 1:1 (Figure 6). Almost the same rate and conversion was observed as with the thevinyl 1:5 acetate ratio, indicating[47]. Therefore, that, atthe this synthesis low aw reaction, essentially at aw no< 0.1 hydrolysis was repeated occurred, for BSLA as was with the a 1-octanol case for 1:5 ratio, indicating that, at this low aw, essentially no hydrolysis occurred, as was the case for Novozymto vinyl acetate 435, as ratio reported of 1:1 (Figure earlier. 6). Overall, Almost these the same differences rate and in conversion performance was at observed altered aasw withcan bethe Novozym 435, as reported earlier. Overall, these differences in performance at altered aw can be ascribed1:5 ratio, to indicating several influences that, at [ 47this,57 ,low58]. Novozymeaw, essentially 435 isno an hydrolysis immobilized occurred, enzyme as and was its highthe activitycase for ascribed to several influences [47,57,58]. Novozyme 435 is an immobilized enzyme and its high canNovozym be linked 435, to as the reported dispersion earlier. of the Overall, enzyme th onese a differences large surface, in performance promoting its at mass altered transfer aw can and be preventingascribedactivity canto particle severalbe linked aggregation. influences to the dispersion In[47,57,58]. contrast, of the Novozyme lyophilizedenzyme on435 enzymesa islarge an surface,immobilized have a promoting reduced enzyme accessibility its massand its transfer of high the individualactivityand preventing can enzymes be linked particle in to the the preparation. aggregation.dispersion Furthermore,of Inthe contenzyrast,me it on isthe wella largelyophilized established surface, promoting thatenzymes immobilized haveits mass a enzymes transferreduced areandaccessibility better preventing protected of the particle againstindividual aggregation. the acetaldehydeenzymes inIn thecont that preparation.rast, is a sidethe productlyophilized Furthermore, of the enzymes acylation it is well have reaction established a reduced [59]. that A accessibilityimmobilized of enzymes the individual are better enzymes protected in the against preparation. the acetaldehyde Furthermore, that it isis awell side established product of that the immobilized enzymes are better protected against the acetaldehyde that is a side product of the

Catalysts 2020, 10, 308 7 of 17 CatalystsCatalysts 2019 2019, 9, ,x 9 FOR, x FOR PEER PEER REVIEW REVIEW 7 of7 17of 17 difference in susceptibility to acetaldehyde induced deactivation might also cause the alterations in acylationacylation reaction reaction [59]. [59]. A differenceA difference in insusceptibili susceptibility tyto toacetaldehyde acetaldehyde induced induced deactivation deactivation might might also also rate between the two pure enzymes. However, similarly, ionization and water clustering can influence causecause the the alterations alterations in inrate rate between between the the two two pure pure enzymes. enzymes. However, However, similarl similarly, ionizationy, ionization and and water water the activity [60,61], leading to these alterations. To demonstrate that the observed effect is general, the clusteringclustering can can influence influence the the activity activity [60,61], [60,61], lead leadinging to tothese these alterations. alterations. To To demonstrate demonstrate that that the the experiments were repeated, but this time with BSLA that was dried by co-lyophilization with a salt to observedobserved effect effect is general,is general, the the experiments experiments were were repe repeated,ated, but but this this time time with with BSLA BSLA that that was was dried dried by by establish the desired aw [62,63]. The enzyme is now in aw different environment and two different aw co-lyophilizationco-lyophilization with with a salta salt to toestablish establish the the desired desired a a[62,63].w [62,63]. The The enzyme enzyme is isnow now in ina differenta different were established, < 0.1 and 0.57.w At < 0.1, very similar results were obtained. Equally, at higher aw, environmentenvironment and and two two different different a awerew were established, established, < 0.1 < 0.1 and and 0.57. 0.57. At At < 0.1, < 0.1, very very similar similar results results were were the ester formation slowed downw as before, but could be restarted by adding additional vinyl acetate obtained.obtained. Equally, Equally, at higherat higher a ,a thew, the ester ester formation formation slowed slowed down down as asbefore, before, but but could could be berestarted restarted by by (Figure7). addingadding additional additional vinyl vinyl acetate acetate (Figure (Figure 7). 7).

Figure 6. Activity of BSLA, toluene at aw < 0.1. Reaction conditions: 0.5–1.2 U of catalyst, 100 mM 1- FigureFigure 6. Activity6. Activity of BSLA,of BSLA, toluene toluene at at aw a

Figure 7. Activity of BSLA co-lyophilized with the appropriate salt, toluene at aw < 0.1 or 0.57. Reaction FigureFigure 7. Activity 7. Activity of BSLAof BSLA co-lyophilized co-lyophilized with with the the appropriate appropriate salt, salt, toluene toluene at a wat< aw0.1 < 0.1 or 0.57.or 0.57. Reaction Reaction conditions:conditions:conditions: 0.5–1.2 0.5–1.2 0.5–1.2 U U ofU of catalyst,of catalyst, catalyst, 100 100 100 mM mM mM 1-octanol, 1-octanol, 1-octanol, 100 100 100 mM mM mM vinyl vinyl vinyl acetate, acetate, acetate, 500 500500 mM mMmM decane decanedecane (ISTD), (ISTD),(ISTD), in in 1-1 indry dry toluene toluene (1 (1mL mL reaction) reaction) at at 30 30 °CC an andd 1000 rpm.rpm. U: µµmolmol butyricbutyric acidacid xmin min .. Blanks-1Blanks werewere dry toluene (1 mL reaction) at 30◦ °C and 1000 rpm. U: µmol butyric acid× x min− . Blanks were performedperformedperformed inin thethein the absenceabsence absence ofof enzyme,enzyme,of enzyme, i.e.,i.e., i.e., inin thethein the presencepres presenceence ofof salt,salt,of salt, andand and showedshowed showed nono conversion.noconversion. conversion. AfterAfter After 88 hh 8 h (480(480(480 min)min) min) anan additionalanadditional additional equivalentequivalent equivalent ofof vinylvinylof vinyl acetateacetate acetate waswas was added.added. added.

ToTo confirm confirm this this activity activity of ofBSLA BSLA (equilibrated (equilibrated via via the the gas gas phase) phase) in indry dry toluene toluene as asa generala general w property,property, the the reaction reaction was was repeated repeated in indry dry meth methyl-t-butylyl-t-butyl ether ether (MTBE) (MTBE) at atthe the same same low low a a

Catalysts 2020, 10, 308 8 of 17

To confirm this activity of BSLA (equilibrated via the gas phase) in dry toluene as a general property, the reaction was repeated in dry methyl-t-butyl ether (MTBE) at the same low aw < 0.1. EnzymesCatalysts 2019 display, 9, x FOR the PEER same REVIEW activity in organic solvents when these have the same aw [64]. Indeed,8 of 17 the BSLA-catalyzed esterification displayed a very similar reaction progress in MTBE and toluene (FigureBSLA-catalyzed8). This confirms esterification the activity displayed of BSLA a very at simila lowr a reactionw, in line progress with the in earlierMTBE and observed toluene catalytic (Figure activity8). This of confirms CALB at the low activity aw [47 of] and BSLA of Rhizomucor at low aw, in miehei line withlipase the at earlier very low observed aw [65]. catalytic activity of CALB at low aw [47] and of Rhizomucor miehei lipase at very low aw [65].

Figure 8. Activity of BSLA, in MTBE and toluene aw < 0.1. Reaction conditions: 0.5–1.2 U of catalyst, Figure 8. Activity of BSLA, in MTBE and toluene aw < 0.1. Reaction conditions: 0.5–1.2 U of catalyst, 100 mM 1-octanol, 500 mM vinyl acetate, 500 mM decane (ISTD), in dry solvent (1 mL reaction) at 30 ◦C 100 mM 1-octanol, 500 mM vinyl acetate, 5001 mM decane (ISTD), in dry solvent (1 mL reaction) at 30 and 1000 rpm. U: µmol butyric acid min− . Blanks were performed in the absence of enzyme and °C and 1000 rpm. U: µmol butyric acid× x min−1. Blanks were performed in the absence of enzyme and showed no conversion. showed no conversion. 3. Discussion 3. Discussion Interestingly, the BioGPS analysis seems to identify features of the BSLA active site which are sharedInterestingly, by other amidase the BioGPS enzymes. analysis In particular, seems to BSLA identi seemsfy features to share of similar the BSLA H-bond active capabilities site which with are amidases,shared by as other evidenced amidase by theenzymes. single-probe In particular, clustering. BSLA The seems possible to share promiscuous similar H-bond amidase capabilities activity of BSLAwith wasamidases, probed as with evidenced an amidase by the activity single-probe assay (Scheme clustering.1)[ 36 ].The This possible revealed promiscuous a complete absenceamidase ofactivity amidase of BSLA activity. was While probed indicative, with an amidase this is not activity conclusive, assay (Scheme as this might1) [36]. alsoThis berevealed due to a substrate complete specificity.absence of Amidases amidase activity. are characterized While indicative, by a developed this is networknot conclusive, of H-bond as this acceptors might andalso donorsbe due as to describedsubstrate inspecificity. previous workAmidases [33]. are The characterized aromatic moiety by a of developed the substrate network molecule of H-bond might thus acceptors prevent and a gooddonors interaction as described with in such previous H-bond work/hydrophilic [33]. The network.aromatic moiety More generally, of the substrate for amidases, molecule the might necessity thus ofprevent a hydrogen a good bond interaction network with that such stabilizes H-bond/hydrophilic the NH hydrogen network. to suppressMore generally, its deprotonation for amidases, was the reportednecessity earlier of a hydrogen [66]. Given bond the network very open that active stabiliz sitees ofthe BSLA, NH hydrogen the minimal to suppress serine hydrolase its deprotonation with an αwas/β hydrolase reported earlier fold, it [66]. is not Given entirely the very surprising open active that this site typeof BSLA, of hydrogen the minimal bond serine network hydrolase has never with beenan α described/β hydrolase for fold, this enzyme.it is not entirely surprising that this type of hydrogen bond network has never beenThe described test of aforw asthis parameter enzyme. for the assignment of a serine hydrolase as lipase gave conclusive results.The BSLA test andof aw CALB as parameter displayed for good the assignment activity at low of a wserine. In line hydrolase with earlier as lipase results, gave the conclusive synthetic catalyticresults. BSLA activity and of CALB CALB displa variesyed depending good activity on the at preparation. low aw. In line Earlier with studiesearlier results, had shown the synthetic that the observedcatalytic syntheticactivity of catalytic CALB varies activity depending competes on with the the preparation. hydrolytic activity—thatEarlier studies is had to say, shown the parallelthat the hydrolysisobserved synthetic of vinyl catalytic acetate [ 47activity]. This competes can lead with to an the apparent hydrolytic decrease activity—that in synthetic is to say, activity, the parallel as is observedhydrolysis for of Novozym vinyl acetate 435 and[47]. BSLA This atcan higher lead atow (Figurean apparent5). This decrease trend has in alreadysynthetic been activity, reported as is forobserved Novozym for 435Novozym [47]. Just 435 like and Novozym BSLA at higher 435 [47 ],aw BSLA (Figure displays 5). This essentially trend hasno already hydrolysis been ofreported vinyl acetatefor Novozym at low a 435w < [47].0.1. ThisJust islike confirmed Novozym in 435 experiments [47], BSLA with displays a ratio essentially of 1:1 of alcohol no hydrolysis to vinyl acetate;of vinyl aacetate similar at rate low of aw synthesis < 0.1. This was is confirmed observed (Figuresin experiments6 and7). with The a fact ratio that of 1:1 free of CALB alcohol displays to vinyl higher acetate; synthetica similar ratesrate atof highersynthesis aw iswas also observed in line with (Figures the literature 6 and 7). [ 47The]. Itfact had that earlier free beenCALB demonstrated displays higher for synthetic rates at higher aw is also in line with the literature [47]. It had earlier been demonstrated for CALB that the ratio of synthesis to hydrolysis depends on the preparation of the enzyme used and that it increases with aw for purified, free CALB [47]. The activity of BSLA at low aw was proven also with a different solvent, MTBE (Figure 8). BioGPS is a complimentary computational tool to investigate the character of an enzyme and delivers a useful input to help us explore the scope of an enzyme more thoroughly. The parameter

Catalysts 2020, 10, 308 9 of 17

CALB that the ratio of synthesis to hydrolysis depends on the preparation of the enzyme used and that it increases with aw for purified, free CALB [47]. The activity of BSLA at low aw was proven also with a different solvent, MTBE (Figure8). BioGPS is a complimentary computational tool to investigate the character of an enzyme and delivers a useful input to help us explore the scope of an enzyme more thoroughly. The parameter aw is an indicative tool to determine whether an enzyme is a lipase or and esterase. Just like the substrate scope, it is not absolute, but is highly indicative. Essentially, a serine hydrolase that is active at low aw is a lipase and not an esterase, while the reverse statement is not valid. Or, as it was recently summarized: “This long-standing and biased question could be compared to the search for differences between and , which implicitly means that one does not consider humans as mammals! Obviously, lipases are a special kind of esterases like humans are a special kind of mammals.” [2].

4. Materials and Methods

4.1. Materials Chemicals and Enzymes 1-propanol, 1-octanol, toluene extra dry, decane, p-nitrophenylbutyrate, p-nitrophenol, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), tributyrin and 2-methyl-2-propanol, butyric acid and caprylic acid were purchased from Sigma-Aldrich (Schnelldorf, Germany) and Acros (Geel, Belgium), and used without previous purification. Vinyl acetate was purchased from Sigma-Aldrich and distilled before use. Novozym 435 (immobilized lipase B from Candida antarctica) was made available by Novozymes (Bagsværd, Denmark). Bovine albumin protein and from chicken egg whites were purchased from Sigma Aldrich. Bradford reagent was purchased from Biorad (Hercules, C.A., USA). Medium and buffer components were purchased from BD, Merck (Darmstadt, Germany) or J.T. Baker (Geel, Belgium). Strains and Plasmids Strains Escherichia coli (E. coli) HB2151 and E. coli HB2151 pCANTAB 5E bsla were kindly provided by Prof. Bauke Dijkstra and Prof. Wim Quax, University of Groningen, the Netherlands. Strains Escherichia coli (E. coli) BL21 (DE3), E. coli TOP10 and plasmid pET22b(+) were utilized for all further work.

4.2. Methods

Cloning pET22bbsla and pET22bcalb The of BSLA (as confirmed by sequencing ID: CP011115.1, range from 292296 to 292841, protein AKCA5803.1) was amplified by PCR from vector pCANTAB 5E BSLA using primers BSLA F: 50-CCTTTCTATGCGGCCCAGC–30 and BSLA + XhoI R: 50-CCGCTCGAGCGCCTTCGTATTCTGG-30. Thereby, restriction site XhoI was introduced for subsequent cloning of BSLA (NcoI, XhoI) into vector pET22b+ (in frame with pelB and His-tag signals). The resulting vector was named pET22bBSLA. The wild type CALB was synthesized by BaseClear (Leiden, The Netherlands). The codon-optimized genes were cloned into pET22b(+) using the NcoI and NotI restriction sites as previously described [67], in order to be in frame with the pelB sequence and a C-terminal His-tag of the plasmid. Expression and Purification of BSLA This protocol was adapted from [24]. A freshly grown colony of E. coli HB 2151 pCANTAB 5E BSLA was used to inoculate a 1 L shake flask containing 100 mL of 2xTY medium (1.6% w/v bactotryptone, 1% w/v bacto yeast extract and 0.5% w/v sodium chloride), ampicillin (100 µg/mL final concentration) and isopropyl-β-d-galactopyranoside (IPTG, 1 mM final concentration). After 16 h at 28 ◦C and 150 rpm (Innova Incubator, Hamburg, Germany) the cells were harvested and washed with Catalysts 2020, 10, 308 10 of 17

10 mM Tris Buffer pH 7.4 and stored at 20 C. The periplasm isolation protocol was adapted from [67] − ◦ and consisted of the resuspension of the overexpressed cells in 1 mL of 10 mM Tris buffer pH 8.0 containing sucrose (25 % w/v), EDTA (2 mM) and lysozyme (0.5 mg/mL). After incubation on ice for 20 min, 250 µL of 10 mM Tris buffer pH 8.0 containing sucrose (20% w/v) and MgCl2 (125 mM) was added. The suspension was centrifuged and the supernatant containing the periplasmic fraction was desalted using a PD10 column (GE, Healthcare, New York, N.Y., USA) to 100 mM potassium phosphate buffer pH 7.4. Afterwards, the solution was shock-frozen with liquid nitrogen and stored at 20 C for future − ◦ biocatalysis applications. BSLA was purified mainly from the media. First, proteins were precipitated by adding 50% v/v saturated ammonium sulphate (2.8 M final concentration) for 5 h at 4 ◦C. After centrifugation, the solid fraction was dissolved in 100 mL of 100 mM potassium phosphate buffer pH 7.4, filtered through a 0.45 µm filter and loaded into a 5 mL His-Trap previously equilibrated column (GE Healthcare) using a NGC chromatography system (BIORAD, Hercules, C.A., USA). The loaded proteins were washed with equilibration buffer potassium phosphate (100 mM, pH 7.4) containing 500 mM NaCl and 20 mM imidazole. The His-tagged BSLA was eluted with a linear gradient from 0–100% potassium phosphate (100 mM, pH 7.4) containing 500 mM NaCl and 500 mM imidazole. The progress of the purification was monitored at 280 nm. Fractions containing the target protein (as confirmed by SDS-PAGE and activity assay, 50–60% of the gradient) were combined, concentrated and desalted with a PD-10 column (GE Healthcare) to potassium phosphate buffer (100 mM, pH 7.4). The purified enzyme (68–148 µg/L medium) was aliquoted (2.5 U/vial, units determined by tributyrin assay) and freeze dried for 16 h, 80 C and stored at 20 C under nitrogen atmosphere. − ◦ − ◦ Protein Sequence of BSLA-His (AKCA5803.1): MAAEHNPVVMVHGIGGASFNFAGIKSYLVSQGWSRDKLYAVDFWDKTGTNYNNGPVLSR FVQKVLDETGAKKVDIVAHSMGGANTLYYIKNLDGGNKVANVVTLGGANRLTTGKALPGTDP NQKILYTSIYSSADMIVMNYLSRLDGARNVQIHGVGHIGLLYSSQVNSLIKEGLNGGGQNTKALEH HHHHH Expression and Purification of CALB An LB-Amp plate (100 µg/mL) was used to freshly grow E. coli BL21 (DE3) pET22bCALB from a 80 C DMSO stock. After incubation at 37 C for 16 h, a single colony was used to inoculate a 5 mL − ◦ ◦ LB-Amp (100 µg/mL) preculture and grown for 8 h at the same temperature. Large-scale expressions were carried out in 0.5 L of ZYM-5052 media (placed in 2 L shake flask), 2% v/v of the preculture was used for inoculation. After 17 h expression at 22 ◦C and 170 rpm, an optical density (600 nm) of approximately 3 was obtained in all cases. Afterwards, cells were spun down, washed with 10 mM potassium phosphate buffer pH 7.4 and stored at 20 C. ZYM-5052 medium [68]: The main cultures − ◦ were grown in ZYM-5052 medium containing 50 mL 50xM (Na HPO 12H O 448 g/L, KH PO 170 2 4· 2 2 4 g/L, NH4Cl 134 g/L, Na2SO4 35.5 g/L), 20 mL 50x5052 (100 g/L α-D-lactose, 250 g/L and 25 g/L glucose dissolved in ddH2O) and 2 mL of MgSO4 solution (1 M in ddH2O) and filled to 1 L with ZY medium (casamino acids 10 g/L—tryptone in this case—yeast extract 5 g/L). Additionally, 0.2 mL of trace element solution was added to the media. The purification of His-tagged CALB was performed from the periplasmic fraction, as described for BSLA in the previous sections. Protein Sequence of CALB-His (Sequence ID: 4K6G_A): MALPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPC WISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMA FAPDYKGTVLAGPLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNS PLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPAN DLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTPAAALEHHHHHH Bradford Assay Catalysts 2020, 10, 308 11 of 17

Total protein concentration was determined using Bradford reagent in a microtiter plate (MTP) reader format (96 well plates) [69]. Properly diluted samples were mixed with Bradford reagent (5x), incubated at room temperature (RT) for 5 min and the absorbance measured at 595 nm (in triplicate). The calibration curve was carried out using bovine serum albumin protein, as is standard. Lipase Activity: Tributyrin Assay A tributyrin assay for determining lipase activity was performed according to the literature [54]. The assay is based on pH change by acid formation when tributyrin is hydrolyzed by the enzyme. p-Nitrophenol was used as pH indicator (colorless at pH 5.5 and yellow at pH 7.5) and the acid concentration was determined by a calibration curve with known amounts of butyric acid (from 0 mM to 40 mM). A negative control was performed by adding buffer instead of an enzyme sample. The substrate consumption (0.8 mM initial concentration) was monitored at 410 nm, 30 ◦C for 15 min, every 38 s by a microtiter plate reader (in 96-well plates, Synergy 2, BioTek, Winooski, V.T., USA). Plates were shaken for 5 s before every read. The different buffers needed for this assay contained 2.5 mM 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.2), CHAPS (to dissolve acids) and β-cyclodextrin (to dissolve acids into the solution, to increase the linearity). The activity was determined in U, which is equivalent to µmol acid formed per minute. The assays were done in triplicate. For performing this assay with immobilized enzymes, a larger scale (3 mL) in glass vials with a magnetic stirrer was applied. These were placed on a stirring platform and, for Novozym 435, samples (120 µL) were taken over time and placed in a 96-well plate. If desired, the assay can also be performed with Trioctanoin. Esterase/Lipase Activity: p-Nitrophenol Assay This protocol was adapted from [53] to an MTP reader equipped for 96 well plates. The enzymatic hydrolysis of p-nitrophenyl butyrate with the concomitant formation of p-nitrophenol was monitored at 405 nm, 37 ◦C and recorded for 30 min. For this, a calibration curve of p-nitrophenol in potassium phosphate buffer (100 mM, pH 7.4) was prepared (levels from 0-500 µM, 200 µL total volume, in triplicate) and control reactions without enzyme extracts were performed. Lyophilized -free extract or pure enzymes were re-dissolved in potassium phosphate buffer (100 mM, pH 7.4) (approximately 20–30 mg/mL) and proper dilutions were added into a preheated potassium phosphate buffer (100 mM, pH 7.4) solution containing 3 mM p-nitrophenyl butyrate. The esterase activity measured was corrected by subtracting the activity observed in the controls (no enzyme). By definition, one unit of enzyme (U) is equivalent to 1 µmol of p-nitrophenol formed per minute. Karl Fischer Titration A Metrohm KF Coulometer Karl Fischer titration setup was used, according to the manufacturer’s instructions, to determine the water content in ppm. Samples (100 µL) were taken from the solvent and injected into the system in duplicate. In general, master mixes of toluene after equilibration with aw < 0.1, aw 0.23 and aw 0.75 contained 20, 120 and 360 ppm respectively. A deviation of 5–10 ppm per sample was observed.

Equilibration of Solvents/Enzymes to the Desired aw All materials, reagents, enzymes and solvents used for the biocatalytic reactions were carefully dried and kept under nitrogen atmosphere with molecular sieves (5 Å) at all times. In all cases, the water content was monitored by Karl Fischer titration and as standard parameter compounds with a water content below 100 ppm were considered dry and suitable for the reaction. Vapor Phase Method Oversaturated salt solutions and activated molecular sieves were used to equilibrate the solvents and enzymes needed in the transesterification reaction with a desired water activity [47–50]. In the case of working with dry systems, a master mix was prepared including solvent, substrates (without vinyl acetate) and internal standard, all components previously dried with activated molecular sieves Catalysts 2020, 10, 308 12 of 17

achieving aw < 0.1. Lyophilized BSLA, Novozym 435 and CALB were dried over silica in desiccators under a vacuum at room temperature (20–25 ◦C) for 24, 48 and 24 h, respectively. In order to achieve higher water activities, the master mix and the enzymes were equilibrated over saturated salt solutions of potassium acetate (KAc) and sodium chloride (NaCl) at 30 ◦C for 48 h, resulting in aw 0.23 and aw 0.75 at 30 ◦C, respectively [51]. As exceptions, BSLA and free CALB were equilibrated for a shorter period of only 24 h. Salt Pairs Method The protocol was adapted from [62]. The enzymes were lyophilized with anhydrous salts (Na2HPO4 or NaAc) in a ratio 1:99 (3 mg pure BSLA or CALB enzyme and 297 mg of the respective salt). For the background reaction (no-enzyme), only lyophilized salts were added. An amount of 10 mg of the co-lyophilized enzyme was added under a nitrogen atmosphere to the previously dried reaction components (except vinyl acetate) and a specific amount of water was introduced under the nitrogen atmosphere. The moles of water added to the reaction mixture were calculated in order to generate the couple of hepta- and dihydrated phosphates in the case of Na2HPO4 (5 moles of water per mol of salt, aw ~ 0.57) and the couple of tri- and anhydrous acetate in the case of NaAc (1.5 moles of water per mol of salt, aw ~ 0.25) [50]. After overnight equilibration, the last substrate was added (freshly distilled and dry vinyl acetate) to begin the reaction. In the case of the dry system, no water was added (aw < 0.1). Transesterification Catalyzed by Lipases in Organic Solvents under Fixed Water Activities The protocol was adapted from [47]. The reaction conditions included substrates 1-propanol, 1-octanol, 2-octanol or benzylacohol (100 mM), vinyl acetate freshly distilled (1 or 5 equiv. in respect to the initial substrate concentration) and decane (ISTD, 500 mM final concentration). A range of 0.5–1 U of purified enzymes, 1 mg of immobilized Novozym 435 were tested as catalysts. Toluene or methyl-t-butyl ether were used as media (1 mL total volume in GC airtight vials). Reactions were carried out for 25 h, 30 ◦C and 1000 rpm (thermoblock Eppendorf, Hamburg, Germany). Negative controls were run for both substrates in absence of enzyme. All reactions were performed in duplicate and monitored over time by gas chromatography. Analytics: GC and GC-MS Gas chromatograph (GC) and gas chromatograph-mass spectrometry (GC-MS) methods were adapted from [47]. Samples were injected in a gas chromatograph (GC-2014, Shimadzu, Kyoto, Japan) equipped with a CP Sil 5 column (50 m 0.53 mm 1.0 um). Injector and detector temperatures × × were set to 340 and 360 ◦C, respectively. The initial column temperature was set to 35 ◦C for 5 min, followed by an increase of 15 ◦C/min up to 60 ◦C for 0.5 min and 15 ◦C/min up to 160 ◦C and hold for 2 min. Finally, a burnout was introduced, 30 ◦C/min up to 325 ◦C. The retention times for 1-propanol, vinyl acetate, toluene, decane, 1-octanol and 1-octylacetate were 1.69, 1.87, 6.64, 10.52, 11.19 and 12.79 min, respectively. To confirm the product’s structure, samples were also injected in a gas chromatograph-mass spectrometer (GC-MS QP2010s, Kyoto, Japan) equipped with a CP Sil 5 (25 m 0.25 mm 0.4 µm). The injector, interface and ion source temperatures were set to 315, 250 and × × 200 ◦C, respectively. The retention times for vinyl acetate, 1-octanol and 1-octylacetate were 1.78, 11.57 and 12.93 min, respectively. Amidase Activity Assay: Hydrolysis of Benzyl Chloroacetamide This protocol was adapted from the literature [36]. The biocatalysis conditions included a total volume 500 µL in a 2 mL Eppendorf tube containing 5 mM benzyl chloroacetamide (stock solution of 500 mM in THF), 100 µg/mL enzyme (as quantified by Bradford assay), in 25 mM potassium phosphate buffer pH 7.0 with 10 % v/v THF. The conversion was carried out for 24 h at 37 ◦C and 500 rpm. Afterwards, the derivatization of 200 µL of reaction mixture was carried out with 50 µL of NBDCl (20 mM in DMSO) for 1 h, at 37 ◦C and 500 rpm. UV detection at 475 nm was performed. Catalysts 2020, 10, 308 13 of 17

BSLA Structure The structure of BSLA was taken from the Protein Data Bank (PDB). The structure downloaded (1R50) was treated by removing all molecules but the protein chain with the software PyMOL. The thus-generated structure was used for visual inspection with PyMOL as well as input for the BioGPS analysis.

4.3. BioGPS Computational Analysis The BioGPS analysis and projection was taken from the previous published work. The BSLA BioGPS analysis was performed using the BioGPS software provided by Molecular Discovery Ltd. (Borehamwood, Hertfordshire, UK) by projecting the enzyme according to its active site properties in the previously performed analysis. The identification of the BSLA active site and the calculation of its properties has been performed as previously described. Specifically, FLAPsite was used for automatic active site identification. The active site was mapped using a GRID approach and the resulting computed properties were considered as electrondensity-like fields centered on each atom, which correspond to the so-called pseudo-molecular interaction fields (pseudo-MIFs). Four different properties were mapped: the active site shape (H probe), H-bond donor properties (O probe), H-bond acceptor capabilities (N1 probe), and hydrophobicity (DRY probe). The magnitude of the interaction of the N1 and O probes also includes, implicitly, information about the charge contribution, as these probes already have a partially positive and negative charge, respectively. The pseudo-MIF points were filtered, by means of a weighted energy-based and space-coverage function, and then used for the generation of quadruplets obtained from all possible combinations of the four pseudo-MIF points. Thus, the BSLA active site was described by a series of quadruplets. Finally, BSLA was projected according to its series of quadruples and scored by the previously performed BioGPS analysis.

5. Conclusions The longstanding question “what differentiates lipases from esterases?” has led to a list of six parameters that are indicative, but not decisive. Here, we have probed BioGPS and aw as parameters to distinguish between lipases and esterases, utilizing the minimal serine hydrolase with an α/β fold, BSLA, as a test enzyme. While BioGPS has been used successfully to address similar questions earlier, it was not indicative in this case. The clear assignment of BSLA as either esterase or lipase was a challenging task. The high catalytic activity of BSLA at low aw clearly demonstrated this serine hydrolase to be a lipase. In future studies, activity at low aw should, therefore, be utilized to support the differentiation of lipases and esterases.

Supplementary Materials: The following file is available online at http://www.mdpi.com/2073-4344/10/3/308/s1, Figure S1: Bio GPS of 43 serine hydrolases, for BSLA the data of pdb 1R50 were utilized: (a) H-bond donor; (b) H-bond acceptor; (c) Hydrophobicity; Table S1: Enzymes utilized for the Bio GPS study with the relevant PDB codes. Author Contributions: Conceptualization, U.H., L.G., P.B. and G.T.; methodology, P.B., V.F. and G.T.; validation, N.v.M., E.A., P.B. and V.F.; formal analysis, P.B., U.H. and L.G.; investigation, N.v.M., E.A., P.B. and V.F.; resources, U.H. and L.G.; data curation, N.v.M., E.A., P.B. and V.F.; writing—original draft preparation, U.H., P.B. and V.F.; writing—review and editing, U.H. and L.G.; visualization, U.H. and L.G.; supervision, P.B., U.H. and L.G.; project administration, U.H. and L.G.; funding acquisition, U.H. and L.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by BE-BASIC, grant number FES-0905 to P.B. and G.T. L.G. received funding from the University of Trieste, FRA 2018. Acknowledgments: Excellent technical support by the technicians of the BOC group and by Linda Otten is gratefully acknowledged. Bauke Dijkstra and Wim Quax, University of Groningen, the Netherlands kindly provided the Escherichia coli (E. coli) HB2151 and E. coli HB2151 pCANTAB 5E bsla. L.G. is grateful to Molecular Discovery Ltd. for providing software access. Conflicts of Interest: The authors declare no conflict of interest. Catalysts 2020, 10, 308 14 of 17

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